HISTORICAL BACKGROUND
The use of oxidizing antiseptics and disinfectants has an interesting development dating back to the late eighteenth century. Because of the relevance of hypohalite and peroxide antiseptics to the present invention, their abbreviated histories are presented. In 1788, the French chemist Berthollet described the disinfecting and bleaching properties of a solution prepared from aqueous alkali and chlorine, and in 1792 a potassium-based preparation of similar composition, eau de Javel, was sold commercially as a disinfectant. In 1820 Labarraque prepared a solution from aqueous sodium carbonate and chlorine. This liqueur de Labarraque was well known for its disinfectant and deodorizer qualities. In 1846 Semmelweis used chloride of lime, a calcium hypochlorite solution, to successfully control the spread of puerperal sepsis, and in 1881 Koch reported his results on the bactericidal action of hypochlorite.
In 1818 Thenard synthesized hydrogen peroxide (H.sub.2 O.sub.2) by reacting dilute acid with barium dioxide to yield a 3 to 4% solution of H.sub.2 O.sub.2 that was relatively unstable. The disinfectant properties of H.sub.2 O.sub.2 were recognized by the mid nineteenth century. "Its application has been advocated for rendering water and milk safe, for disinfection of sewage; it has been applied in medicine, surgery, dentistry, hair-dressing etc" (Heinemann, 1913, J.A.M.A. 60: 1603-1606). However, its antiseptic capacity is relatively poor in comparison with hypochlorites.
The antiseptic action of dyes was also known and used prior to and during the First World War. In 1900 Raab reported that the dye acridine killed living cells (i.e., paramecia) only in the presence of light (Z. Biol. 39: 524 et seq.), and in 1905 Jodlbauer and von Tappeiner demonstrated that O.sub.2 was required for the dye-sensitized photokilling of bacteria (Deut.Arch.Klin.Med. 82: 520-546). Dye-sensitized, O.sub.2 -dependent photooxidation and photooxygenation reactivity is commonly referred to as photodynamic activity (Blum, 1941, Photodynamic Action and Diseases Caused by Light, Reinhold, New York). Dyes, such as flavine and brilliant green, were effective as antiseptic agents even when employed at relatively high dilutions in serous medium. Unfortunately, in addition to their potent antimicrobial action, these dyes also produce host damage, i.e., leukocyte killing (Fleming, 1919, Brit.J.Surg. 7: 99-129).
Research in the area of antiseptic action was accelerated by the First World War. During this period the previously described potency of hypochlorite-based antiseptics (Andrewes and Orton, 1904, Zentrabl.Bakteriol.(Orig.A) 35: 811-816) was firmly established, and preparations, such as Eusol (Smith et al., 1915, Brit.Med.J. 2: 129-136) and Dakin's solution (Dakin, 1915, Brit.Med.J. 2: 318-320) supplanted the initially favored carbolic acid and iodine antiseptics.
Alexander Fleming's 1919 Hunterian lecture (supra), entitled, "The Action of Chemical and Physiological Antiseptics in a Septic Wound" provides an excellent exposition of the subject of antisepsis that is relevant to this day. Fleming described two schools of thought regarding the treatment of wounds: (1) the physiological school which directed "their efforts to aiding the natural protective agencies of the body against infection", and (2) the antiseptic school which directed their efforts to killing the wound microbes with chemical agents.
The physiologic school maintained that the greatest protection against infection was obtained by aiding the physiological agencies: (1) blood and humoral defense mechanisms, and (2) phagocytic leukocytes. It was known that leukocytes collected in the walls and emigrate into the cavity of the wound, ultimately forming the cellular elements of pus. Fleming noted that the phagocytic leukocytes of "fresh pus" exert potent antimicrobial effect, but that "stale pus" (i.e., pus from an unopened furuncle), as well as heat-treated or antiseptic-treated "fresh pus", lack microbe killing capacity.
The Nonspecific Nature of Antiseptic Treatment:
The basic problem of the chemical approach to antisepsis is that chemical antiseptics react non-specifically. "Disinfection is a chemical reaction in which the reactive agent acts not only on bacteria but upon the media in which they are found" (Dakin, 1915, Brit.Med.J. 2: 809-810). Antiseptic solutions produce maximum microbe killing when the organisms are suspended in an aqueous medium, but germicidal action is greatly decreased by competitive reaction with the organic matter present in serous fluid or blood.
Antiseptics can non-specifically react with and inhibit normal immunophysiologic defense mechanisms. Germicidal concentrations of antiseptics inhibit the antimicrobial function of phagocytic leukocytes. "The leukocytes are more sensitive to the action of chemical antiseptics than are the bacteria, and, in view of this, it is unlikely that any of these antiseptics have the power of penetrating into the tissues and destroying the bacteria without first killing the tissues themselves. The natural antiseptic powers of the pus are done away with, but the microbes are not completely destroyed, and those which are left are allowed to grow unhindered until such time as fresh pus-cells can emigrate to keep them in check. A consideration of the leucocidal property of antiseptics will show us that certain antiseptics are suitable for washing of a wound, while others are bad. If we desire, therefore, an antiseptic solution with which to wash out a wound, we should choose one which loses its antileucocytic power rapidly and which exercises its antiseptic action very quickly. We then have the washing effect of the fluid without doing much damage to the wound. One great advantage of eusol and Dakin's solution is that they disappear as active chemical agents in a few minutes and do not have any lasting deleterious effect on the leukocytes" (Fleming, 1919).
Mechanism of Action:
Many of the early workers believed that hypochlorite microbicidal action was dependent on the nascent oxygen liberated as a product of hypochlorous acid autoprotolysis, and that the liberated oxygen combined with the unsaturated components in the cell protoplasm to effect killing. This view was challenged early in this century by Dakin. "It has been repeatedly stated that the antiseptic action of hypochlorous acid was due to the liberation of oxygen. I have been unable to find any evidence to support this statement." He went on to propose a more direct chlorination mechanism. "It appears that when hypochlorous acid and hypochlorites act upon organic matter of bacterial or other origin some of the (NH) groups of the proteins are converted into (NCl) groups. The products thus formed--belonging to the group of chloramines--I have found to possess approximately the same antiseptic action as the original hypochlorite, and it appears more probable that the antiseptic action of the hypochlorites is conditioned by the formation of these chloramines rather than by any decomposition with liberation of oxygen" (Dakin, 1915). Furthermore, it was known that "oxygen from sources other than chlorine does not kill bacteria as readily as does the amount of chlorine theoretically necessary to yield an equivalent amount of nascent oxygen" (Mercer and Somers, 1957, Adv.Food Res. 7: 129-160).
Dakin's position on the direct microbicidal action of chlorine, which persists to the present, is also problematic. "Experimental proof is lacking also for other hypotheses advanced to explain the bactericidal action of chlorine. These include suggestions that bacterial proteins are precipitated by chlorine; that cell membranes are altered by chlorine to allow diffusion of cell contents; and that cell membranes are mechanically disrupted by chlorine" (Mercer and Somers, 1957). Chlorine-binding to bacteria is remarkably low at pH 6.5 and is doubled by raising the pH to 8.2 (Friberg, 1956, Acta Pathol.Microbiol.Scand. 38: 135-144). On the other hand, the bactericidal and virucidal capacity of hypochlorite is increased by acidity, i.e., by towering the pH (Butterfield et al., 1943, Publ.Health Reports 58: 1837-1866; Friberg and Hammarstrom, 1956, Acta Pathol. Microbiol.Scand. 38: 127-134). As such, chlorine-binding is inversely related to chlorine-dependent killing.
Organic chloramine preparations, e.g. chloramine-T, also serve as antiseptic agents, but paradoxically, the microbicidal action of these chloramines is concluded to result in whole or in large part from the hypochlorous acid formed from chloramine hydrolysis (Leech, 1923, J.Am.Pharm. Assoc. 12: 592-602). Chloramine bactericidal action "may be due in whole or in part to the hypochlorous acid formed in accordance with the hydrolysis and ionization equilibria" (Marks et al., 1945, J.Bacteriol. 49: 299-305). The greater stability afforded by the slower hydrolysis of chloramines slows germicidal action.
Hypochlorite exerts a bactericidal action at concentrations of 0.2 to 2.0 ppm, (i.e., 4 to 40 nmol per ml). The high potency of such a "trace" concentration strongly suggest that microbicidal action results from the inhibition of an essential enzyme or enzymes (Green, 1941, Adv.Enzymol. 1: 177-198). Evidence has been presented that hypochlorous acid inhibits various sulfhydryl enzymes and that inhibition of glucose metabolism is proportional to bacterial killing (Knox et al., 1948, J.Bacteriol. 55: 451-458).
The literature with regard to the mechanism of H.sub.2 O.sub.2 action is somewhat incomplete. However, the overall consensus is that "H.sub.2 O.sub.2 in spite of its high oxidation-reduction potential is as sluggish an oxidizing agent as molecular oxygen and in fact a large number of oxidations attributed to this substance have been found, on careful examination, to be due to free radical formation which occurs on addition of catalytic amounts of Fe.sup.++ or Cu.sup.++ " (Guzman-Barron et al., 1952, Arch.Biochem.Biophys. 41: 188-202). This view expresses the consensus conclusion of several studies (Yoshpe-Purer and Eylan, 1968, Health Lab.Sci. 5: 233-238; Miller, 1969, J.Bacteriol. 98: 949-955).
Various dyes have also been used as antiseptics. Photodynamic action results when a dye (.sup.1 Dye), i.e., a singlet multiplicity sensitizer molecule, absorbs a photon and is promoted to its singlet excited state (.sup.1 Dye*). If .sup.1 Dye* decays back to its .sup.1 Dye ground state by photon emission, the phenomenon of fluorescence is observed without photodynamic action. In order to serve as a photodynamic sensitizer the .sup.1 Dye* must undergo intersystem crossing (ISC), i.e., change in spin multiplicity, to yield the metastable triplet excited state of the dye (.sup.3 Dye*) in relatively high quantum yield (Gollnick, 1968, Advan.Photochem. 6: 1-122): EQU .sup.1 Dye---h.nu..fwdarw..sup.1 Dye*---ISC.fwdarw..sup.3 Dye*(1)
Sensitizers absorb light ranging from the near ultraviolet throughout the visible to include the near infrared. This absorption is responsible for the color properties of the "dye". The wavelength of light (i.e., the energy of the photon) required for dye excitation is defined by the absorption spectrum of the dye.
The .sup.3 Dye* state is relatively long-lived and as such, can react with other molecules. Photodynamic reactions can be divided into two main classes depending on the reactivity of .sup.3 Dye* (Schenck and Koch, 1960, Z.Electrochem. 64: 170-177). In Type I reactions the excited triplet sensitizer undergoes direct redox transfer with another molecule. Sensitizers for Type I reactions typically are readily oxidized or reduced . EQU .sup.3 Dye*+.sup.1 SubH.fwdarw..sup.2 Dye+.sup.2 Sub. (2)
In equation (2), the triplet sensitizer serves as a univalent oxidant and is reduced to its doublet state (.sup.2 Dye), and the singlet multiplicity substrate (.sup.1 SubH) is oxidized to a doublet multiplicity, free radical .sup.2 Sub. state. In a analogous fashion, a reducing .sup.3 Dye* may serve as a radical reductant. The .sup.2 Dye product of reaction (2) can react with ground state O.sub.2, a triplet multiplicity diradical molecule (.sup.3 O.sub.2), to yield the doublet multiplicity hydrodioxylic acid radical (.sup.2.O.sub.2 H) or its conjugate base the superoxide anion (.sup.2.O.sub.2.sup.-) and regenerate the singlet ground state of the dye: EQU .sup.2 Dye+.sup.3 O.sub.2 .fwdarw..sup.1 Dye+.sup.2.O.sub.2 H(or .sup.2.O.sub.2.sup.-) (3)
Under neutral to acid conditions these products of oxygen reduction undergo doublet-doublet (i.e., radical-radical) annihilation to yield H.sub.2 O.sub.2 : EQU .sup.2.O.sub.2 H+.sup.2.O.sub.2.sup.- +H.sup.+ .fwdarw..sup.1 H.sub.2 O.sub.2 +.sup.1 O.sub.2 ( 4)
If the reaction is by direct annihilation spin conservation will be maintained, and as such, singlet molecular oxygen (.sup.1 O.sub.2) can also be produced (Khan, 1970, Science 168: 476-477).
In Type II reactions the excited triplet sensitizer interacts directly with triplet (ground state) .sup.3 O.sub.2. Reaction involves the spin-balanced transfer of excitation energy from .sup.3 Dye* to .sup.3 O.sub.2 yielding the ground state .sup.1 Dye and singlet molecular oxygen (.sup.1 O.sub.2) as products (Kautsky, 1939, Trans.Faraday Soc. 35: 216-219): EQU .sup.3 Dye*+.sup.3 O.sub.2 --.sup.1 DyeO.sub.2 .fwdarw..sup.1 Dye+.sup.1 O.sub.2 ( 5)
Reaction (5) is very fast and is the most common Type II pathway. However, if .sup.3 Dye* is sufficiently reducing, direct univalent electron transfer to O.sub.2 may occur: EQU .sup.3 Dye*+.sup.3 O.sub.2 --.sup.1 DyeO.sub.2 .fwdarw..sup.2 Dye.sup.+ +.sup.2.O.sub.2.sup.- ( 6)
Radical annihilation can proceed to yield H.sub.2 O.sub.2 as described by reaction (4). In considering these reaction pathways it should be appreciated that reaction (5) is favored over reaction (6) by over two orders of magnitude (Kasche and Lindqvist, 1965, Photochem. Photobiol. 4: 923-933).
Microbial killing by dyes could result from the reaction of the .sup.3 Dye* itself or its Type I and Type II reaction products, i.e., .sup.2.O.sub.2.sup.-, H.sub.2 O.sub.2, and especially .sup.1 O.sub.2, with microbial proteins, nucleic acids, unsaturated lipids, et cetera (Spikes and Livingston, 1969, Adv.Rad.Biol. 3: 29-121).
.sup.1 O.sub.2, a broad spectrum electrophilic oxygenating agent, can inhibit enzymes by destroying amino acids essential to catalytic activity. The rate constants (k.sub.r, in M.sup.-1 sec.sup.-1) for the reaction of .sup.1 O.sub.2 with tryptophan, histidine, and methionine range from 2*10.sup.7 to 9*10.sup.7 (Matheson and Lee, 1979, Photochem.Photobiol. 29: 879-881; Kraljic and Sharpatyi, 1978, Photochem.Photobiol. 28: 583-586). If generated in close proximity to a target microbe, a "trace" quantity of .sup.1 O.sub.2 could effectively inhibit enzymes required for microbe metabolism. Unsaturated lipids, nucleic acids and other electron dense biological molecules are also reactive with .sup.1 O.sub.2. The dioxygenation of such essential cellular components might also play a part in microbicidal action.
The Continuing Problem:
The following essential points can be distilled from the preceding material. First, high potency chemical antiseptics are typically oxidizing agents, e.g., HOCl. These oxidizing and oxygenating agents are capable of microbicidal action in "trace" quantities, and probably exert their effects via inhibition of enzymes essential for metabolism (Green, 1941).
Second, the antimicrobial potency of such antiseptics is compromised by their nonspecific reactivity. Damage is not limited to the target microbe. As pointed out by Fleming, host cells are generally more susceptible than microbes to toxic action of antiseptics.
An ideal antiseptic agent would exert potent reactivity against a broad range of pathogenic microbes including fungi with minimum toxicity to host cells. In keeping with the principles of the physiological school of wound care, an antiseptic should aid or augment the natural protective agencies of the body against infection.
To a limited extent, these requirements are met by certain antibiotics. The selective bactericidal action of antibiotics is based on differences between prokaryotic and eukaryotic cells with regard to protein synthesis, nucleic acid replication, and the presence or composition of the cell wall. Antibiotics can, in effect, selectively poison certain bacteria, i.e., prokaryotic organisms, without poisoning the eukaryotic host cells. However, the broad spectrum action of antibiotics can have detrimental effects on the bacteria that make up the normal flora of the host. The bacteria of the normal flora serve as a barrier to the growth of pathogenic organisms, and as such, antibiotic destruction of the normal flora provides an opportunity for the growth of more pathogenic bacteria. Antibiotic-associated pseudomembranous colitis results from the overgrowth of pathogens, i.e., Clostridium difficile and rarely Staphylococcus aureus, following antibiotic destruction of normal flora.
In addition, yeast and fungi are eukaryotic microbes, and as such, are essentially unaffected by antibiotics. Consequently, yeast overgrowth and infections can follow antibiotic treatment of bacterial infections.
Antibiotics can also exert direct toxic effects. These detrimental effects can result from the direct action of the drug on host cells and tissue, e.g., the nephrotoxicity of antibiotics.
As is readily apparent from the foregoing, there is a long felt need for new and improved antiseptics which have reactivity against a broad range of pathogenic microbes, but exhibit a minimum of activity toward host cells and normal flora.